We have developed a new in vivo mouse model to image single cancer-cell dynamics of metastasis to the lung in real-time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re-inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony formation were imaged in real-time. This new technology makes it possible to observe real-time monitoring of cancer-cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley-Liss, Inc.